Targeting FGFR4 protein degradation targeting chimera as well as preparation method and application thereof

Abstract

The invention discloses a protein degradation targeting chimera targeting FGFR4 as well as a preparation method and application thereof, and relates to the technical field of biological medicines. Wherein the protein degradation targeting chimera has a structure as shown in a formula (V1), and is formed by coupling a covalent inhibitor ligand targeting FGFR4 and an E3 ubiquitin ligase ligand through a linker; according to the preparation method, an intermediate is constructed through multi-step organic synthesis, and finally a target compound is obtained through an amide condensation reaction. The compound can be specifically combined with FGFR4 protein and induce ubiquitination degradation of the FGFR4 protein, and has a remarkable curative effect in preparation of drugs for treating FGFR4-mediated tumors. In the development of the FGFR4 targeted protein hydrolysis chimera, a lead compound with potential is provided for the first time, and the problem that the FGFR4 targeted protein hydrolysis chimera becomes available from nothing is solved.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a protein degradation-targeting chimera that targets FGFR4, its preparation method, and its application. Background Technology

[0002] Fibroblast growth factor receptor 4 (FGFR4) is a receptor tyrosine kinase belonging to the fibroblast growth factor receptor family. It plays a crucial role in maintaining metabolic homeostasis and tissue development. Studies have shown that abnormal activation of the FGF19 / FGFR4 signaling pathway, caused by FGFR4 gene amplification, point mutation, or gene fusion, is closely related to the development and progression of various malignant tumors, particularly hepatocellular carcinoma, breast cancer, and colorectal cancer. Therefore, targeting FGFR4 has become an important clinical strategy for treating these dependent cancers.

[0003] Currently, treatments targeting FGFR4 primarily focus on developing small molecule inhibitors, such as BLU9931, H3B-6527, and FGF401. These compounds inhibit FGFR4 activity by competitively binding to the ATP-binding site of the kinase domain, and some have already entered clinical trials. However, as clinical research progresses, the limitations of traditional small molecule inhibitors are becoming increasingly apparent. First, long-term use of tyrosine kinase inhibitors often leads to acquired resistance, which is usually related to secondary mutations in the target protein. Second, to achieve sufficient inhibitory effects, high dosages are typically required, which may cause serious toxic side effects and safety issues. Furthermore, for certain non-enzymatic scaffold functions, simple activity inhibition is often insufficient to completely block tumor progression.

[0004] In recent years, protein degradation-targeted chimeric technology has emerged as a novel drug development strategy. This strategy recruits E3 ubiquitin ligases to the vicinity of target proteins, utilizing the ubiquitin-proteasome system to achieve targeted degradation. Compared to traditional inhibitors, protein degradation strategies possess catalytic cycling characteristics, enabling complete clearance of target proteins at lower concentrations, potentially overcoming drug resistance mutations and improving targeting selectivity. While numerous studies have been conducted on compounds targeting other targets, a mature approach that simultaneously achieves efficient degradation, stable covalent binding, and significant induction of tumor cell apoptosis remains lacking in the FGFR4 field. Therefore, developing a novel, highly efficient FGFR4 protein degrader with good pharmacological activity is of significant clinical and research value for improving the treatment of FGFR4-mediated diseases such as hepatocellular carcinoma. Summary of the Invention

[0005] This invention provides a protein degradation-targeting chimera that targets FGFR4, its preparation method, and its application. In the development of FGFR4-targeting protein hydrolysis chimeras, this invention provides a promising lead compound for the first time, solving the problem of developing FGFR4 protein-targeting chimeras from scratch.

[0006] In a first aspect, embodiments of the present invention provide a protein degradation targeting chimera that targets FGFR4, the protein degradation targeting chimera having a structure as shown in formula (V1): Its chemical name is: (2S,4R)-1-((R)-2-(2-(4-(3-acryloylamino-4-((6-(3-(2,6-dichloro-3,5-dimethoxyphenyl)-1-methylurea)pyrimidin-4-yl)amino)phenyl)piperazin-1-yl)acetamyl)-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide.

[0007] Preferably, it further includes a pharmaceutically acceptable salt of the protein degradation-targeting chimera, the pharmaceutically acceptable salt being selected from hydrochloride, sulfate, phosphate, methanesulfonate, citrate, maleate, fumarate, or tartrate.

[0008] Secondly, embodiments of the present invention provide a method for preparing the protein degradation targeting chimera that targets FGFR4 as described in the foregoing embodiments, comprising the following steps: The intermediate compound shown in formula (11) was subjected to an amide condensation reaction with (2S,4R)-1-((S)-2-amino-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide in the presence of a condensing agent and a base to prepare the protein degradation targeting chimera shown in formula (V1); The intermediate compound shown in formula (11) has the following structure: Its chemical name is: 2-[4-(3-acrylamido-4-((6-(3-(2,6-dichloro-3,5-dimethoxyphenyl)-1-methylureido)pyrimidin-4-yl)amino)phenyl)piperazin-1-yl]acetic acid.

[0009] Preferably, the condensing agent is selected from one or more of HATU, EDCI, DCC or T3P; the base is selected from one or more of DIEA, triethylamine, pyridine or 4-dimethylaminopyridine.

[0010] Preferably, it further includes the step of preparing the intermediate compound shown in formula (11): The intermediate compound shown in formula (9) was reacted with tert-butyl bromoacetate under alkaline conditions to prepare the intermediate compound shown in formula (10). Subsequently, the intermediate compound shown in formula (10) was hydrolyzed under acidic conditions to obtain the intermediate compound shown in formula (11). The structural formulas of equations (9) and (10) are shown below: .

[0011] Thirdly, embodiments of the present invention propose the use of the protein degradation targeting chimera that targets FGFR4 as described in the foregoing embodiments in the preparation of a medicament for treating tumors, wherein the tumor is an FGFR4-mediated tumor.

[0012] Preferably, the FGFR4-mediated tumor is selected from hepatocellular carcinoma.

[0013] Preferably, the drug induces tumor cell apoptosis by downregulating the expression level of FGFR4 protein in tumor cells.

[0014] Preferably, the tumor cells are hepatocellular carcinoma JHH-7 cells that overexpress FGFR4.

[0015] Beneficial Effects: The FGFR4-targeting protein degradation chimera provided by this invention achieves highly efficient and selective degradation of the target protein through innovative structural design. Compound V1 possesses excellent covalent binding ability, quantitatively forming a 1:1 covalent addition product with FGFR4 protein; the half-maximal degradation concentration in hepatocellular carcinoma JHH-7 cells is as low as 0.84 μM, with a maximum degradation rate of 80%, and it exhibits excellent rapid onset and long-lasting degradation characteristics, with its effect lasting up to 72 hours. Furthermore, this compound can significantly induce tumor cell apoptosis in a concentration-dependent manner, effectively overcoming the limitations of traditional inhibitors such as easy drug resistance and insufficient potency, providing a highly efficient, long-acting, and clinically promising candidate for FGFR4-mediated malignant tumor treatment. Attached Figure Description

[0016] Various other advantages and benefits will become apparent to those skilled in the art upon reading the following detailed description of preferred embodiments. The accompanying drawings are for illustrative purposes only and are not intended to limit the invention. Furthermore, the same reference numerals denote the same parts throughout the drawings. In the drawings: Figure 1 This is an Intact-MS image showing the complete protein mass spectrometry (Intact-MS) detection results of the covalent binding of compound V1 of the present invention to FGFR4 protein.

[0017] Figure 2The figure shows the detection results of the degradation effect of compound V1 of the present invention on FGFR4 protein in JHH-7 cells after treatment at different concentrations for 48 hours.

[0018] Figure 3 The figure shows the detection results of the degradation effect of compound V1 of the present invention on FGFR4 protein in JHH-7 cells at different treatment times at a concentration of 1.25 μM.

[0019] Figure 4 This is a flow cytometry result of the compound V1 of this invention inducing apoptosis in JHH-7 cells.

[0020] Figure 5 This is a flowchart of the preparation method of compound V1 of the present invention. Detailed Implementation

[0021] To more clearly illustrate the technical solutions in the embodiments of the invention or the prior art, the invention will be briefly introduced below in conjunction with the accompanying drawings and descriptions of the embodiments or the prior art. Obviously, the following description of the structure of the drawings is only some embodiments of the invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. It should be noted that the description of these embodiments is for the purpose of helping to understand the invention, but does not constitute a limitation on the invention.

[0022] In the specific implementation of this invention, unless otherwise specified, all chemical reagents and solvents were purchased from commercial suppliers and were not further purified before use. Anhydrous solvents were prepared by standard drying methods or purchased directly as anhydrous grade products. Proton nuclear magnetic resonance (NMR) spectroscopy... 1 ¹H NMR and carbon-13 nuclear magnetic resonance (CMR) spectra, 13 The C10 NMR data were determined using an NMR spectrometer, and the solvents used included deuterated dimethyl sulfoxide (DMSO). d 6 DMSO- d 6The chemical shifts were expressed in ppm, and the coupling constants in Hz. Mass spectrometry analysis was performed using liquid chromatography-mass spectrometry (LC-MS) in electrospray ionization (ESI) mode. High-resolution mass spectrometry (HRMS) data were obtained using a high-resolution mass spectrometer. The reaction process was monitored by thin-layer chromatography (TLC) using pre-coated silica gel GF254 glass plates observed under UV light, or by colorimetric reagents such as iodine vapor, potassium permanganate solution, or ninhydrin solution. Column chromatography separation and purification used 200-300 mesh silica gel as the stationary phase, and the eluent was selected based on TLC monitoring results. High-performance liquid chromatography (HPLC) analysis is performed using a system equipped with a C18 reversed-phase column.

[0023] Example 1 This embodiment details the synthetic route and specific operational procedures for the compound shown in formula (V1), namely (2S,4R)-1-((R)-2-(2-(4-(3-acryloylamino-4-((6-(3-(2,6-dichloro-3,5-dimethoxyphenyl)-1-methylurea)pyrimidin-4-yl)amino)phenyl)piperazin-1-yl)acetamyl)-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide. The specific preparation method of compound V1 of this invention is as follows: Figure 5 As shown. Figure 5 The process clearly demonstrates that the preparation route starts from the starting materials and constructs the target molecule through multiple orderly chemical transformations. The flowchart details the key intermediate structures and transformation logic of each step of the reaction. In particular, it clearly indicates the intermediate with the structure shown in formula (9) generated after the introduction of the acrylamide warhead, and the intermediate with the structure shown in formula (10) generated after the subsequent substitution reaction. Finally, the protein degradation targeting chimera shown in formula (V1) is assembled through hydrolysis and condensation reactions.

[0024] Synthesis of Intermediate 2, N-(4-bromo-2-nitrophenyl)acetamide: In a clean, dry 250 mL single-necked round-bottom flask, the starting material 4-bromo-2-nitroaniline was added, followed by acetic anhydride and 50 mL of glacial acetic acid. Magnetic stirring was started, and after the solid was completely dissolved, a catalytic amount of methanesulfonic acid was slowly added dropwise to the reaction system at room temperature. After the addition was complete, the reaction flask was placed in an oil bath and heated to 100°C, and the reaction was stirred at this temperature for 3 hours. Heating was stopped after the starting material spot was confirmed to have disappeared by TLC monitoring, and the reaction solution was allowed to cool naturally to room temperature. The cooled reaction solution was slowly poured into a beaker containing 150 mL of ice water while stirring vigorously; a large amount of yellow solid precipitated. Stirring continued for 30 minutes to ensure complete precipitation. The mixture was filtered under reduced pressure through a Buchner funnel, and the filter cake was washed three times with cold water to remove residual acid and impurities. The filter cake was collected and placed in a vacuum drying oven, where it was dried overnight at 50°C to obtain a yellow solid intermediate 2, with a yield of 83.6%. The proton NMR spectrum data of this intermediate are as follows: 1 H NMR (400 MHz, DMSO- d 6 ) δ 10.29 (s, 1H), 8.08 (d, J = 2.3 Hz, 1H), 7.86 - 7.83 (m, 1H), 7.54 - 7.51 (m, 1H), 2.02 (s, 3H).

[0025] Synthesis of Intermediate 3, tert-butyl 4-(4-acetamido-3-nitrophenyl)piperazine-1-carboxylate: Intermediate 2, N-Boc-piperazine, and cesium carbonate were added sequentially to a 250 mL three-necked flask equipped with a reflux condenser. 80 mL of anhydrous toluene was added as the reaction solvent. Subsequently, the catalysts 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene (Xantphos) and tris(dibenzylideneacetone)dipalladium (Pd2(dba)3) were added. The reaction system was evacuated three times and purged with high-purity nitrogen to ensure an oxygen-free environment. Under nitrogen protection, the reaction mixture was heated to 100°C and refluxed with vigorous stirring for 4 hours. The reaction progress was monitored by TLC. After the reaction was complete, the mixture was cooled to room temperature and filtered through a funnel lined with diatomaceous earth. The filter cake was washed with a small amount of ethyl acetate. The filtrate was transferred to a separatory funnel, and 200 mL of water was added to separate the organic layer. The aqueous layer was extracted three times with ethyl acetate. All organic phases were combined, washed once with saturated brine, and dried over anhydrous sodium sulfate. The drying agent was removed by filtration, and the filtrate was concentrated to dryness under reduced pressure. The crude product was purified by silica gel column chromatography using a gradient elution of petroleum ether and ethyl acetate to obtain a red solid intermediate 3 in 54.3% yield. The 1H NMR spectrum of this intermediate is as follows: 1 H NMR (400 MHz, DMSO- d 6 ) δ 9.90 (s, 1H), 7.35 - 7.32 (m,2H), 7.26 - 7.13 (m, 1H), 3.43 - 3.40 (m, 4H), 3.14 -3.12 (m, 4H), 1.95 (s,3H), 1.38 (s, 9H).

[0026] Synthesis of Intermediate 4, tert-butyl 4-(4-amino-3-nitrophenyl)piperazine-1-carboxylate: Intermediate 3 was placed in a 250 mL round-bottom flask and dissolved in 50 mL of ethanol. Potassium hydroxide was dissolved in 40 mL of water to prepare a solution, which was then added to the ethanol solution at room temperature. The reaction mixture was stirred in a 60°C oil bath for 2 hours to remove the acetyl protecting group. After the reaction was complete, most of the ethanol was removed by rotary evaporation under reduced pressure. The residue was diluted with 150 mL of ethyl acetate, transferred to a separatory funnel, and washed twice with water. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a black solid, intermediate 4. This product had high purity and could be used directly in subsequent reactions without further purification, with a yield of 92.1%. The 1H NMR spectrum of this intermediate is as follows:1 H NMR (400 MHz, DMSO- d 6 ) δ 7.32 - 7.29 (m, 1H),7.27 - 7.24(m, H), 7.20 (s, 2H), 6.94 (d, J = 9.2 Hz, 1H), 3.42 - 3.39 (m, 4H), 2.91 - 2.88 (m, 4H), 1.38 (s, 9H).

[0027] Synthesis of Intermediate 5, tert-butyl-4-[4-((6-(methylamino)pyrimidin-4-yl)amino)-3-nitrophenyl]piperazine-1-carboxylate: Intermediate 4 was dissolved in 100 mL of anhydrous toluene in a dry reaction flask. 6-Chloro-N-methylpyrimidin-4-amine, sodium tert-butoxide, ligand 2-dicyclohexylphosphino-2',4',6'-triisopropylbiphenyl (Xphos), and catalyst Pd2(dba)3 were added sequentially. Under a nitrogen atmosphere, the mixture was heated to 100°C and stirred for 4 hours. After the reaction was complete, it was cooled to room temperature. The reaction mixture was filtered, and the filter cake was washed with ethyl acetate. The combined filtrates were added to 100 mL of water, and the layers were separated. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography using petroleum ether and ethyl acetate as eluents to obtain intermediate 5, a red powder, in 43.1% yield. The 1H NMR spectrum of this intermediate is as follows: 1 H NMR (400MHz, DMSO- d 6 ) δ 8.93 (s, 1H), 7.92 (s, 1H), 7.61 - 7.58 (m, 1H), 7.38 (d, J =2.9 Hz, 1H), 7.30 - 7.27 (m, 1H), 6.86 - 6.83 (m, 1H), 5.67 (s, 1H), 3.44 -3.42 (m, 4H), 3.12 - 3.09 (m, 4H), 2.69 (d, J = 4.7 Hz, 3H), 1.38 (s, 9H).

[0028] Synthesis of Intermediate 6, tert-butyl 4-[4-((6-(3-(2,6-dichloro-3,5-dimethoxyphenyl)-1-methylureido)pyrimidin-4-yl)amino)-3-nitrophenyl]piperazine-1-carboxylate: In the isocyanate preparation stage, 2,6-dichloro-3,5-dimethoxyaniline and triphosgene were dissolved in 50 mL of anhydrous tetrahydrofuran (THF) in a fume hood. Triethylamine was slowly added dropwise at room temperature. After the addition was complete, the mixture was heated under reflux and stirred for 30 minutes to ensure complete reaction. The reaction solution was concentrated to dryness under reduced pressure to obtain a white solid, which is the in-situ generated isocyanate intermediate. In the urea formation stage, 60 mL of anhydrous toluene was added to the above white solid, followed by intermediate 5. The reaction mixture was heated to 100°C and stirred for 3 hours. After the reaction was complete, insoluble matter was removed by filtration. The filtrate was diluted with 200 mL of water and extracted with ethyl acetate. The organic layers were combined, washed with saturated brine, dried, and concentrated. The crude product was purified by recrystallization in ethyl acetate and petroleum ether to obtain intermediate 6 in 79.5% yield. The 1H NMR spectrum of this intermediate is as follows: 1 H NMR (400MHz, DMSO- d 6 ) δ 11.86 (s, 1H), 9.58 (s, 1H), 8.26 (s, 1H), 7.50 - 7.49 (m,1H), 7.42 (d, J = 2.9 Hz, 1H), 7.31 - 7.28 (m, 1H), 6.86 (s, 1H), 6.52 (m, 1H), 3.90 (s, 6H), 3.45 - 3.42 (m, 4H), 3.30 (s, 3H), 3.17 - 3.15 (m, 4H), 1.39 (s,9H).

[0029] Synthesis of intermediate 7, tert-butyl 4-[3-amino-4-((6-(3-(2,6-dichloro-3,5-dimethoxyphenyl)-1-methylureido)pyrimidin-4-yl)amino)phenyl]piperazine-1-carboxylate: Intermediate 6 was dissolved in a mixture of tetrahydrofuran and methanol in a 100 mL reaction flask. Palladium on carbon (Pd / C) was added as a reducing agent. The reaction system was placed under a hydrogen atmosphere and stirred at 60°C for 2 hours. After the reaction was confirmed to be complete by TLC, the catalyst was removed by diatomaceous earth filtration, and the filtrate was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography with dichloromethane and methanol as eluents to obtain the title compound 7 in 78.3% yield. The 1H NMR data of this intermediate are as follows: 1H NMR (400 MHz, DMSO- d 6 )δ 12.12 (s, 1H), 8.61 (s, 1H), 8.25 (s, 1H), 6.90 (d, J = 8.6 Hz, 1H), 6.85(s,1H), 6.32 (d, J = 2.7 Hz, 1H), 6.20 - 6.17 (m, 1H), 6.01 (s, 1H), 4.75 (s,2H), 3.89 (s, 6H), 3.42 - 3.39 (m, 4H), 3.17 (s, 3H), 3.01 - 2.97 (m, 4H), 1.38 (s,9H).

[0030] Synthesis of Intermediates 8 and 9: Intermediate 7 and N-methylpyrrolidone (NMP) were added to a 50 mL round-bottom flask. The mixture was cooled to -10°C, and acryloyl chloride was slowly added dropwise. The reaction was stirred at this low temperature for 4 hours. After the reaction was complete, the reaction solution was diluted with ethyl acetate and water, and separated. The organic phase was washed several times with saturated brine to remove NMP, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain intermediate 8 in 85.7% yield. The 1H NMR spectrum of intermediate 8 is as follows: 1 H NMR (400 MHz, DMSO- d 6 ) δ 12.06 (s, 1H), 9.61 (s,1H), 8.74 (s, 1H), 8.30 (s, 1H), 7.31 - 7.28 (m, 2H), 6.84 (s, 1H), 6.80 -6.77 (m, 1H), 6.50 - 6.44 (m, 1H), 6.23 - 6.18 (m, 2H), 5.71 - 5.67 (m, 1H), 3.89 (s, 6H), 3.47 -3.43 (m, 4H), 3.20 (s, 3H), 3.10 -3.02 (m, 4H), 1.39 (s,9H). Subsequently, intermediate 8 was dissolved in dichloromethane (DCM), and trifluoroacetic acid (TFA) was added. The reaction was stirred at room temperature for 1 to 2 hours to remove the Boc protecting group. After the reaction was completed as monitored by TLC, the solution was concentrated to dryness under reduced pressure to obtain the trifluoroacetate salt of intermediate 9 as shown in formula (9). Given the active chemical properties of the amino group on the piperazine ring of this compound, which is prone to deterioration, it was immediately added to the next step of the reaction without further purification.

[0031] Synthesis of Intermediates 10 and 11: Freshly prepared intermediate 9 was dissolved in anhydrous N,N-dimethylformamide (DMF). Anhydrous potassium carbonate was added as a base, followed by tert-butyl bromoacetate. The reaction mixture was stirred at room temperature for 2 hours. After the reaction was complete, the reaction solution was poured into 40 mL of water and extracted with ethyl acetate. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography with dichloromethane and methanol as eluents to obtain intermediate 10 of formula (10) in a yield of 65.5%. The 1H NMR spectrum of intermediate 10 is as follows: 1 H NMR (400 MHz, DMSO- d 6 ) δ 12.03 (s, 1H), 9.56 (s, 1H), 8.67 (s,1H), 8.28 (s, 1H), 7.29 - 7.21 (m, 2H),6.85 (s, 1H), 6.78 - 6.75 (m, 1H),6.48 - 6.41 (m, 1H), 6.21 - 6.16 (m, 2H), 5.73 - 5.66 (m, 1H), 3.89 (s, 6H), 3.18 (s, 3H), 3.12 (s, 2H), 3.11 - 3.06 (m,4H), 2.65 - 2.57 (m, 4H), 1.39 (s,9H). Subsequently, intermediate 10 was dissolved in an appropriate amount of dichloromethane, and trifluoroacetic acid was added. The mixture was stirred at room temperature to hydrolyze the tert-butyl ester group. After the reaction was complete, the solvent and excess acid were removed by concentration under reduced pressure to obtain carboxylic acid intermediate 11, which was used directly in the final condensation step.

[0032] Synthesis of final product V1: Intermediate 11, the E3 ligase ligand fragment (2S,4R)-1-((S)-2-amino-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide, and the condensing agent 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) were dissolved in 6 mL of anhydrous DMF. N,N-Diisopropylethylamine (DIEA) was added with stirring to adjust the pH to alkaline. The reaction mixture was stirred at room temperature for 1 hour. The reaction was monitored by TLC until complete. For post-treatment, water and ethyl acetate were added to the reaction solution, and the mixture was separated. The organic layer was washed sequentially with water, saturated sodium bicarbonate solution, and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography using a gradient elution of dichloromethane and methanol to obtain a white powder, compound V1, with an overall yield of 19.6% and an HPLC purity of 99.7%. Its high-resolution mass spectrometry data are as follows: HRMS (ESI): m / z [M + H]+ Calculated value C 52 H 63 Cl2N 12 O8S: 1085.3988; Measured value: 1085.3990. The proton NMR data are as follows: 1 H NMR (400 MHz, DMSO- d 6 ) δ 12.08 (s, 1H), 9.59 (s, 1H), 8.94 (s, 1H), 8.74 (s, 1H), 8.43 (d, J = 7.6 Hz, 1H), 8.29 (s, 1H), 7.76 (d, J = 9.6 Hz, 1H), 7.40 - 7.25 (m, 6H), 6.85 (s, 1H), 6.82 - 6.79 (m,1H), 6.49 - 6.43 (m, 1H), 6.22 - 6.17 (m, 2H), 5.79 - 5.60 (m,1H), 5.12 -5.11 (m, 1H), 4.89 - 4.83 (m, 1H), 4.50 - 4.47 (d, J= 9.7 Hz, 1H), 4.44 - 4.38(m, 1H), 4.30 - 4.18 (m, 1H), 3.89 (s, 6H), 3.61 - 3.50(s, 2H), 3.18 - 3.12(m, 7H), 3.08 - 2.97 (m, 2H), 2.64 - 2.57 (m, 4H), 2.41 (s, 3H), 2.05 - 2.00(m, 1H), 1.76 - 1.69 (m, 1H), 1.33 (d, J = 7.0 Hz, 3H), 0.92 (s, 9H). The carbon spectrum data are: 13 C NMR (151 MHz, DMSO- d 6 ) δ 170.93, 164.09, 163.18, 160.00, 158.52,156.26, 154.71, 153.28, 151.92, 148.20,145.23, 135.00, 132.42, 132.23,131.57, 130.13, 129.28, 127.36, 127.19, 126.77, 116.65, 113.07, 97.13, 69.23,58.99, 57.19, 56.98, 48.24, 38.23, 36.20, 32.12,32.00, 31.42, 29.89, 28.94, 26.77, 22.94, 22.53, 16.45, 14.43, 11.71.

[0033] Example 2 This embodiment aims to prepare different salt forms of compound V1 to optimize its physicochemical properties, such as solubility or stability.

[0034] Preparation of compound V1 hydrochloride: Compound V1 prepared in Example 1 was dissolved in 5 mL of anhydrous ethyl acetate. A 1.0 M solution of ethyl acetate hydrochloride was slowly added dropwise with stirring in an ice bath at 0°C. A white precipitate was observed to form during the addition. After the addition was complete, stirring was continued at 0°C for 30 minutes. The precipitate was collected by filtration, and the filter cake was washed with a small amount of cold ethyl acetate and dried under vacuum to obtain the hydrochloride of compound V1 as a white solid. The solubility of this hydrochloride in water was significantly increased compared to that of the free base.

[0035] Preparation of sulfate of compound V1: Compound V1 was dissolved in ethanol. A sulfuric acid ethanol solution was slowly added dropwise at room temperature. The mixture was stirred at room temperature for 2 hours, and a precipitate formed. The precipitate was collected by filtration, washed with cold ethanol, and dried under vacuum to obtain the sulfate of compound V1.

[0036] Preparation of phosphate of compound V1: Compound V1 was suspended in methanol. Phosphoric acid solution was added with stirring until the solid was completely dissolved. The solution was concentrated to dryness, and isopropanol was added and the mixture was ground to obtain phosphate of compound V1 as an amorphous powder.

[0037] Preparation of compound V1 methanesulfonate: Compound V1 was dissolved in 5 mL of acetone. Methanesulfonic acid was added at room temperature. The mixture was stirred at room temperature for 1 hour. The solvent was then removed by concentration under reduced pressure, and the mixture was ground with a small amount of diethyl ether to obtain the methanesulfonate of compound V1 as an off-white powder.

[0038] Preparation of citrate of compound V1: Compound V1 and anhydrous citric acid were added to 10 mL of methanol and heated under reflux until the solid was completely dissolved. The solution was cooled to room temperature and allowed to stand overnight to crystallize. The crystals were collected by filtration and dried to obtain citrate of compound V1.

[0039] Preparation of maleate of compound V1: Compound V1 was dissolved in ethyl acetate and heated to 40°C. Maleic acid solution was added. The mixture was cooled to room temperature and stirred overnight. The precipitated solid was collected by filtration and dried to obtain maleate of compound V1.

[0040] Preparation of compound V1 fumarate: Compound V1 and fumaric acid were added to a mixed solvent of ethanol and water, and the mixture was heated under reflux to dissolve. After cooling and crystallization, the solution was filtered and dried to obtain the fumarate of compound V1.

[0041] Preparation of compound V1 tartrate: Compound V1 was dissolved in methanol. L-tartaric acid was added and stirred until dissolved. Diethyl ether was slowly added as an antisolvent until turbidity appeared. Crystallization was allowed to occur, filtered, and dried to obtain the tartrate of compound V1.

[0042] Example 3 This embodiment uses complete protein mass spectrometry (Intact-MS) technology to verify whether compound V1 can form the expected covalent bond with FGFR4 protein.

[0043] Experimental materials: Recombinant human fibroblast growth factor receptor 4 (FGFR4) kinase domain protein was purchased from a commercial reagent supplier. Compound V1 was prepared in Example 1 and dissolved in dimethyl sulfoxide (DMSO) to prepare a 10 mM stock solution.

[0044] Experimental Methods: Recombinant human FGFR4 protein was diluted to an appropriate concentration. Two experimental groups were set up: the control group consisted of FGFR4 protein with DMSO solvent, and the experimental group consisted of FGFR4 protein with compound V1. The mixture was incubated at room temperature for 2 hours. After incubation, unbound small molecules were removed from the sample by passing it through a desalting column. The sample was then analyzed using liquid chromatography-mass spectrometry (LC-MS).

[0045] Experimental results: such as Figure 1 As shown, mass spectrometry analysis revealed that in the control group, a molecular ion peak of the FGFR4 protein was detected, with a deconvolutioned molecular weight of 34664.0 Da. In the experimental group, the original protein peak was not detected; instead, a new main peak was observed with a molecular weight of 35750.0 Da. The calculated mass shift was 1086.0 Da. This shift value is highly consistent with the theoretical molecular weight of compound V1.

[0046] Example 4 This embodiment evaluated the degradation ability of compound V1 on endogenous FGFR4 protein in different hepatocellular carcinoma cell lines, covering both dose-dependent and time-dependent studies.

[0047] Experimental materials: The cell line was human hepatocellular carcinoma cell line JHH-7. Antibodies used included anti-FGFR4 antibody, anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody, or anti-Tubulin antibody.

[0048] Dose-dependent assay: JHH-7 cells were seeded in 6-well plates. After cell adhesion, the medium was replaced with fresh medium containing different concentrations of compound V1. A concentration gradient was set up. An equal volume of DMSO was added to the control group. After co-incubation with the drug for 48 hours, cells were collected. Total protein was extracted using radioimmunoprecipitation assay (RIPA) lysis buffer. Protein concentration was determined by the bicinchoninic acid (BCA) method. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed, followed by membrane transfer, blocking, and incubation with primary and secondary antibodies. Finally, enhanced chemiluminescence (ECL) imaging was used. Gray-scale analysis of protein bands was performed using image analysis software to calculate the expression level of FGFR4 relative to the internal control protein and the degradation rate.

[0049] Dose-dependent experimental results: The results are as follows Figure 2 As shown, compound V1 significantly induced the degradation of FGFR4 protein in JHH-7 cells. With increasing drug concentration, the FGFR4 protein level gradually decreased. Software fitting showed that the half-maximal degradation concentration (MCC) in JHH-7 cells after 48 hours of treatment was 0.84 μmol, with a maximum degradation rate of 80%. Notably, at extremely high concentrations, a hook-like effect characteristic of protein degradation-targeting chimeras (PROTACs) was observed, indicating a slight recovery in degradation efficiency, consistent with the kinetics of ternary complex formation.

[0050] Time-dependent experimental methods and results: JHH-7 cells were treated with 1.25 μmol of compound V1, and cell samples were collected at different time points for immunoblotting analysis. Results are as follows: Figure 3 As shown, a significant decrease in FGFR4 protein levels was observed 0.5 hours after drug administration, indicating that V1 has a rapid onset of action. Furthermore, this degradation effect can be maintained for up to 72 hours, demonstrating that V1 possesses long-lasting degradation properties.

[0051] Example 5 This embodiment investigates the effect of compound V1 on apoptosis in JHH-7 liver cancer cells overexpressing FGFR4.

[0052] Experimental Methods: JHH-7 cells were seeded in 6-well plates and cultured overnight. Blank control, positive control, low-dose compound V1 group, and high-dose compound V1 group were added for treatment, respectively. After 48 hours of treatment, cells were collected. Cells were washed twice with pre-chilled phosphate-buffered saline (PBS). Staining was performed using a double staining kit with fluorescein isothiocyanate (FITC) and propidium iodide (PI). Immediately after staining, flow cytometry was used for analysis.

[0053] Experimental results: such as Figure 4 As shown, flow cytometry results indicated that control group cells were mainly concentrated in the normal viable cell region, with a low total apoptosis rate. The positive control group showed significant apoptosis, verifying that blocking the FGFR4 pathway can induce apoptosis in this cell line. The total apoptosis rate in the low-dose compound V1 group significantly increased to 38.56%. The total apoptosis rate in the high-dose compound V1 group further increased to 47.31%.

[0054] Finally, it should be noted that although embodiments of the present invention have been shown and described, those skilled in the art will understand that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents.

Claims

1. A protein degradation-targeting chimera that targets FGFR4, characterized in that, The protein degradation targeting chimera has the structure shown in formula (V1): Its chemical name is: (2S,4R)-1-((R)-2-(2-(4-(3-acryloylamino-4-((6-(3-(2,6-dichloro-3,5-dimethoxyphenyl)-1-methylurea)pyrimidin-4-yl)amino)phenyl)piperazin-1-yl)acetamyl)-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide.

2. The protein degradation targeting chimera targeting FGFR4 according to claim 1, characterized in that, It also includes a pharmaceutically acceptable salt of the protein degradation-targeting chimera, wherein the pharmaceutically acceptable salt is selected from hydrochloride, sulfate, phosphate, methanesulfonate, citrate, maleate, fumarate, or tartrate.

3. A method for preparing the protein degradation-targeting chimera targeting FGFR4 as described in claim 1, characterized in that, Includes the following steps: The intermediate compound shown in formula (11) was subjected to an amide condensation reaction with (2S,4R)-1-((S)-2-amino-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide in the presence of a condensing agent and a base to prepare the protein degradation targeting chimera shown in formula (V1); The intermediate compound represented by formula (11) has the following structure: Its chemical name is: 2-[4-(3-acrylamido-4-((6-(3-(2,6-dichloro-3,5-dimethoxyphenyl)-1-methylureido)pyrimidin-4-yl)amino)phenyl)piperazin-1-yl]acetic acid.

4. The method according to claim 3, characterized in that, The condensing agent is selected from one or more of HATU, EDCI, DCC or T3P; the base is selected from one or more of DIEA, triethylamine, pyridine or 4-dimethylaminopyridine.

5. The method according to claim 3, characterized in that, It also includes the step of preparing the intermediate compound represented by formula (11): The intermediate compound shown in formula (9) was reacted with tert-butyl bromoacetate under alkaline conditions to prepare the intermediate compound shown in formula (10). Subsequently, the intermediate compound shown in formula (10) was hydrolyzed under acidic conditions to obtain the intermediate compound shown in formula (11). The structural formulas of equations (9) and (10) are shown below: 。 6. The use of the FGFR4-targeting protein degradation-targeting chimera of claim 1 in the preparation of a medicament for treating tumors, characterized in that, The tumor is an FGFR4-mediated tumor.

7. The use according to claim 6, characterized in that, The FGFR4-mediated tumors were selected from hepatocellular carcinoma.

8. The use according to claim 7, characterized in that, The drug induces tumor cell apoptosis by downregulating the expression level of FGFR4 protein in tumor cells.

9. The use according to claim 8, characterized in that, The tumor cells were JHH-7 hepatocellular carcinoma cells that overexpressed FGFR4.

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